Human leukocyte elastase is a serine protease that is widely dispersed throughout the body and plays an important role in degrading foreign material as part of the body's normal inflammatory response. Prolonged exposure to high levels of HLE has been associated with the onset of such disease states as pulmonary emphysema, adult respiratory distress syndrome (ARDS), chronic bronchitis, cystic fibrosis, rheumatoid arthritis, and atherosclerosis. See, e.g., A. Janoff, Am. Rev. Respir. Dis. 132:417-433 (1985); J. C. Taylor et al., Pulmonary Emphysema and Proteolysis, New York: Academic Press, 1987; C. -B. Laurell et al., Scand. J. Clin. Lab. Invest. 15:132-140 (1963); T. A. Merritt et al., J. Clin. Invest. 72:656-666 (1983); R. A. Stockley et al., Ann. N.Y. Acad. Sci. 624:257-266 (1991); A. H. Jackson et al., J. Respir. Dis. 65:114-124 (1984); L. Eskerot et al., Adv. Exp. Med. Biol. 167:335-344 (1984); and A. Janoff, Annu. Rev. Med. 36:207-216 (1985). The excessive levels of HLE associated with the aforementioned diseases are believed to be the result of insufficient production of its natural inhibitor, .alpha.1-protease inhibitor (.alpha.1-PI).
The protease-antiprotease imbalance theory for HLE-related diseases originated from the observation that people inherently deficient in .alpha.1-PI develop an accelerated form of emphysema. C.-B. Laurell et al., supra. Environmental oxidants, such as cigarette smoke, have been shown to be able to oxidize a methionine residue of .alpha.1-PI that is essential for inhibitory activity (H. Carp et al., Proc. Natl. Acad. Sci. 770:2041-2045 (1982). The resulting oxidized .alpha.1-PI is orders of magnitude less potent that .alpha.1-PI. The chemotactic properties of HLE result in the recruitment of more neutrophils to the site of inflammation. The initial imbalance is amplified by the release of more HLE by the newly recruited neutrophils.
A rational approach to the therapeutic treatment of HLE-related diseases is to reestablish the protease-antiprotease imbalance using exogenously produced inhibitors to HLE. Researchers have developed the proper cloning vectors and have expressed the natural inhibitor .alpha.1-PI using recombinant technologies (H. P. Schnebli, Ann. N.Y. Acad. Sci. 624:212-218 (1991), and augmentation therapy using .alpha.1-PI is being evaluated clinically. This approach has merit in that the therapeutic agent is a naturally occurring substance and is the natural inhibitor for HLE; however, the cost and route of administration used for peptides like .alpha.1-PI make this therapy less than desirable.
Several approaches have been investigated for finding low-molecular-weight mechanism-based inhibitors to HLE. Mechanism-based inhibitors are compounds that bind to a specific class of enzyme (e.g., serine proteases) and are processed like the normal substrates; however, during processing the inhibitors react with active site residues and are either released slowly or not at all from the enzymatic cleft. Mechanism-based inactivators, i.e., inhibitors which act irreversibly, are distinctly different from alkylating agents in that inactivators are completely nonreactive until enzymatic processing. The mechanism of HLE action is well understood and as shown in Scheme 1, consists of five major steps. Following initial formation of a Michealis complex, the substrate carboxyl is attacked by the active site serine (Ser-195) to form a tetrahedral intermediate that collapses to form an acylated HLE intermediate (C-terminal cleaved product released). Hydrolysis regenerates the enzyme, releasing the N-terminal cleaved product. In general, mechanism-based inhibitors to HLE either form very stable tetrahedral intermediates or act as alternate substrates for the enzyme, while mechanism-based inactivators of HLE form very stable acylated HLE intermediates that are resistant to hydrolysis. ##STR2##
Efforts to develop mechanism-based inhibitors can be divided into two rational design strategies: those directed to development of peptide-derived inhibitors, on the one hand, and those directed to development of non-peptide inhibitors, on the other. In general, peptide-derived inhibitors are designed to resemble the natural substrate sequence and act to form stable tetrahedral intermediates. Examples of peptide-derived inhibitors include boronic acid, aldehyde, .alpha.-diketone and .alpha.-diketone and .alpha.-ketoester, .alpha.-fluoro-ketone, and .alpha.-ketobenzoxazole derivatives (D. H. Kin der et al., J. Med. Chem. 28:1917-1925 (1985); C. H. Hassal et al., FEBS Lett. 183:201-205 (1985); S. Mehdi et al., Biochem. Biophys. Res. Commun. 166:201-205 (1990); R. A. Wildonger et al., "The in vitro and in vivo inhibition of human leukocyte elastase by .alpha.,.alpha.-difluoro-.beta.-ketoamides", in Eleventh American Symposium Abstracts, poster 87, presented at University of California, San Diego, Jul. 9-14, 1989; J. W. Skiles et al., J. Med. Chem. 35:641-662 (1992); and P. D. Edwards, J. Am. Chem. Soc. 114:1854-1863 (1992)). A number of nonpeptidic inhibitors have been discovered that are specific for serine proteases and show some selectivity for HLE. These compounds generally act to inactivate the enzyme by forming stable acylated enzyme intermediates. Examples of nonpeptidic mechanism-based inactivators of HLE include ynenol lactones, isocoumarins, cephalosporins, azetidinones, and benzoxazinones. (Copp, L. J. et al., Biochemistry 26:169-178 (1987); Harper, J. W. et al., Biochemistry 24:7200-7213 (1985); Hernandez, M. A. et al., J. Med. Chem. 35:1121-1129 (1992); Harper, J. W. et al., Biochemistry 24:1831-1841 (1985); Finke, P. E. et al., J. Med. Chem. 35:3731-3744 (1992); Shah, S. K. et al., J. Med. Chem. 35:3745-3754 (1992); Krantz A. et al., J. Med. Chem. 33:464-479 (1990)).
Most of the reported HLE mechanism-based inhibitors, however, lack plasma solubility, protease stability, and/or enzyme specificity which makes them unsuitable for pharmaceutical development. Accordingly, there remains a need to discover and develop new therapeutic agents that will be effective in treating emphysema and other HLE-related diseases.
The present invention is directed to a novel class of HLE inhibitors which do not have the above-identified disadvantages of the compounds of the prior art. The inhibitors are benzoxazinones substituted at the 6-position as will be discussed in detail below. The effectiveness of these compounds is quite surprising in view of the teaching in the art that substitution at R.sub.6 is highly unfavorable and gives rise to compounds which would not be effective HLE inhibitors (see, e.g., A. Krantz et al., J. Med. Chem. 33:464-479 (1990)). The novel compounds are potent and specific inhibitors of HLE, and are designed to have greater bioavailability than previous benzoxazinone inhibitors.